Since the first coronavirus infecting chickens was reported in 1937, about 15 coronaviruses belonging to the Coronaviridae family have so far been found to infect humans and animals including cattle, pigs, cats, dogs, birds, rodents, etc. Infections of animals like cattle, pigs, chickens, horses, etc. have major economical impacts on the animal husbandry industry. Particularly, porcine epidemic diarrhea, transmissible gastro-enteritis, and infectious bronchitis, caused by various animal coronaviruses, spread rapidly. Infection of infant animals is often associated with a high mortality rate. Currently, there are no effective therapeutic agents for coronavirus infection, which is economically damaging the animal husbandry industry. Regarding the coronaviruses infecting humans, human coronavirus is known to be responsible for 10-30% of all common colds in adults. SARS-CoV, emerged in the Guangdong province of China in November 2002, and spread to 26 countries worldwide including Hong Kong, Singapore, Vietnam, Canada, USA, etc. It became a worldwide health concern, with a significant impact on the economy as well. SARS is a respiratory disease caused by SARS-CoV, which is a coronavirus variant and is associated with symptoms such as fever, cough, respiratory distress, atypical pneumonia and the like. The latent period of SARS-CoV is about 2-7 days and 10-20% of patients suffer from acute respiratory distress syndrome with a death rate of 7-8%. According to World Health Organization (WHO) report in 2003, approximately 8,000 patients were found to be infected with SARS-CoV and approximately 770 of them died during the SARS outbreak in 2003. Except for several suspected SARS cases, additional infection has not been reported since April 2004. However, WHO and Center for Disease Control and Prevention (CDC) have been keeping a close watch on SARS as there are possibilities of re-emergence.
SARS-CoV belongs to the Coronavirus genus of the Coronaviridae family and has approximately 29.7 kb of positive-stranded RNA genome with cap-structure at 5′-end and poly(A) tail at 3′-end. SARS-CoV has 14 open reading frames (ORF) and 9 intergenic sequences flanked by 5′ and 3′ untranslated regions (UTRs) which are essential cis-acting elements for viral replication. A total of eight subgenomic RNAs are additionally transcribed from intergenic sequences to produce eight structural and accessory proteins. The ORF1 is composed of ORF1a and ORF1b, and the latter is translated by −1 ribosomal frameshift as a polyprotein, which is further processed by two viral proteases to generate a total of 28 viral proteins (Snijder et al., J. Mol. Biol. 331:991-1004).
It is known that RNA-dependent RNA polymerase (RdRp) plays a pivotal role in viral replication. The RdRp, probably with the help of various cellular proteins, initiates viral replication by recognizing cis-acting elements of viral RNA genome in infected cells. SARS-CoV nsp12 is encoded as a first protein in ORF1b and generated by processing of the above described polyprotein by 3C-like proteinase (nsp5). SARS-CoV nsp12 has the SDD motif that is common to coronavirus RdRps, suggesting that replication of SARS-CoV RNA genome might be mediated by nsp12 RdRp that is not present in human cells and is encoded by the viral genome.
In the absence of vaccines and therapeutic agents for various coronaviruses, development of functional RdRps is desperately needed to screen for inhibitors that can be used as antiviral agents. Moreover, antiviral drug screening in cell culture systems using a highly infectious SARS-CoV with a high mortality rate has various practical application limitations. Therefore, in vitro drug screening systems need to be developed. Indeed, tremendous efforts have been made to establish such systems using purified recombinant viral RdRps of SARS-CoV or other coronaviruses belonging to the Coronavirus genus and Torovirus genus in the Cornaviridae family. However, successful cases have not yet been reported. Even with the mouse hepatitis virus for which the coronavirus replication mechanism has been studied extensively since the 1980's, recombinant RdRp has not yet been developed and so in vitro RdRp assay systems have not been established. This is likely due to (1) low expression level of RdRp, (2) its insolubility when over-expressed, and (3) inability of purified RdRps to recognize and transcribe viral RNA templates. Recently, glutathione S-transferase-fused SARS-CoV RdRp (nsp12) was expressed and purified from E. coli. The recombinant protein was expressed mainly in insoluble form and cleaved into several fragments. Moreover, the authors did not demonstrate that the fusion RdRp protein, yet partially purified with its several cleaved forms, was able to copy RNA templates derived from the viral RNA genome (Cheng et al., Virology 335:165-176). Similarly, a purified recombinant RdRp of equine arteritis virus, which belongs to the Arterivirus genus in Nidovirale order, was not shown to be able to copy viral genome-derived RNA templates (Beerens et al., J. Virol. 81:8384-8395). In vitro replication systems for coronaviruses could be useful in studying viral RNA replication mechanisms, identifying target sites of antiviral agents by mapping of cis-acting elements, and screening for inhibitors against RdRp. Functional recombinant RdRp is the key element of the in vitro replication system. However, previous studies including the works described above have not yet established a robust in vitro replication system using a soluble, purified functional RdRp capable of utilizing the 3′-end RNA regions on both plus- and minus-strands of viral RNA, as templates.